Carbon dots (CDs) were synthesized via a simple method and applied in bioimaging. The CDs display excitation-independent fluorescence emission and exhibit upconversion PL, providing further tissue penetration and greater sensitivity as the effect of the autofluorescence of biological bodies is minimized and cellular imaging. This is the first time that excitation-independent carbon dots is prepared via a truly facile approach without the need of external heating or additional energy input. These results indicate that the CDs have excellent potential to be applied in biomedical and clinical analysis.